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United States Patent |
5,554,523
|
Reddy
,   et al.
|
September 10, 1996
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Nucleic acid sequences encoding human leucine-zipper protein-kinase
Abstract
A novel protein kinase, leucine-zipper protein kinase, 668 amino acids in
length is provided by the present invention. This protein kinase is
localized to the human brain. Nucleic acid sequences encoding the protein
kinase are also provided.
Inventors:
|
Reddy; Usharani (North Wales, PA);
Pleasure; David (Wynnewood, PA)
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Assignee:
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Children's Hospital of Philadelphia (Philadelphia, PA)
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Appl. No.:
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205018 |
Filed:
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March 1, 1994 |
Current U.S. Class: |
435/194; 435/252.3; 435/320.1; 536/23.2 |
Intern'l Class: |
C12N 001/20; C12N 015/00; C12N 009/12 |
Field of Search: |
435/194,320.1,252.3
536/23.2
|
References Cited
Other References
Asano et al., "Domains Responsible for the Differntial Targeting of Glucose
Transporter Isoforms", J. Biol. Chem. 267: 19636-19641 (1992).
Forman et al., "A Domain Containing Leucine-Zipper-Like Motifs mediate
novel In Vivo Interactions Between the Thyroid hormone and Retinoic Acid
Receptors", Molecular Endocrinology 3: 1610-1626 (1989).
Pleasure et al., "Pure, Postmitotic, Polarized Human Neurons Derived from
NTera 2 Cells Provide a System for Expressing Exogenous Proteins in
Terminally Differentiated Neurons", J. Neuroscience 12: 1802-1814 (1992).
Wernet et al., "the cDNA of The Two Isoforms of Bovine cGMP-Dependent
Protein Kinase",FEBS251: 191-196 (1989).
Younkin et al., "Inducible Expression of Neuronal Glutamate Receptor
Channels in the NT2 Human Cell Line", Proc. Natl. Acad. Sci. USA 90:
2174-2178 (1993).
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Primary Examiner: Wax; Robert A.
Assistant Examiner: Hendricks; Keith D.
Attorney, Agent or Firm: Woodcock Washburn Kurtz Mackiewicz & Norris
Goverment Interests
REFERENCE TO GOVERNMENT GRANTS
The work present herein was supported in part by National Institute of
Health grants NS08075, NS25044 and NS31102. The United States government
may have certain rights in the invention.
Claims
What is claimed is:
1. cDNA coding for a human leucine-zipper protein kinass.
2. cDNA encoding a protein which is at least 85% homologous to a protein
encoded by the nucleic acid sequence set forth in SEQ ID NO: 1.
3. A construct comprising a vector and the cDNA of claim 2.
4. The construct of claim 3 further comprising a promoter operably linked
to said cDNA.
5. Recombinant host cells transformed with cDNA of claim 2 whereby said
host cells express leucine-zipper protein kinass.
6. A method of producing leucine-zipper protein kinass which comprises
culturing recombinant host cells wherein said host cells are transformed
with cDNA encoding a protein which is at least 85% homologous to a protein
encoded by SEQ ID NO:1 operably linked to regulatory control sequences
which effect the expression of said coding sequence in said transformed
host cells and isolating said leucine-zipper protein kinase produced by
said host cells.
Description
FIELD OF THE INVENTION
This invention is directed to a novel protein-kinase, nucleic acid
sequences encoding the same and methods related thereto.
BACKGROUND OF THE INVENTION
Protein kinases regulate various cellular responses to changing
environmental conditions. Protein kinases fall into two general classes:
those protein kinases that transfer phosphate to serine or threonine and
those proteins that transfer phosphate to tyrosine (Krebs and Beavo, Annu.
Rev. Blochem 48: 923-959 (1979)). A few protein kinases, such as weel, now
appear to be capable of phosphorylating both ser/threonine and tyrosine
(Lindberg et al., Trends Biochem Sci 17: 114-119 (1992)). Phosphorylation
is of particular significance in controlling mitogenesis and cellular
differentiation. Receptors for a number of polypeptide growth factors are
transmembrane tyrosine kinases (Yarden and Ullrich, Annu. Rev. Biochem 57:
443-478 (1988)), which in turn stimulate serine/threonine kinases such as
protein kinase C, MAP kinase and p74.sup.raf (Hunter et al., Nature 311:
480-483 (1984); Morrison et al., Cell 58: 649-657 (1989); Rossomondo et
al., Proc. Natl. Acad. Sci. USA 86: 6940-6943 (1989)) .
Protein kinases, and especially the overexpression thereof, have been found
to be linked to hyperproliferation of cells and metastasis. Many protein
kinases were first identified as the products of oncogenes and still
constitute the largest family of known oncogenes. Lindberg and Hunter,
Mol. and Cell. Biol., 10(11): 6316-6324 (1990).
Mutations of genes encoding members of the protein kinase family which are
involved in the regulation of neuroblastic proliferation, differentiation
and survival play a role in the etiology of human central nervous system
tumors. Thus, it is highly desirable to gain a greater understanding of
this class of proteins, as well as to use such greater understanding to
limit or inhibit the effects that these proteins have on cellular
hyperproliferation.
SUMMARY OF THE INVENTION
There is provided by the present invention a cDNA sequence encoding a novel
protein kinase, leucine-zipper protein kinase (zpk), and the protein
encoded thereby.
There are provided by the present invention recombinant constructs encoding
leucine-zipper protein kinase.
There are provided by the present invention novel methods of use and
diagnosis for leucine-zipper protein kinase and cDNA coding for
leucine-zipper protein kinase.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F. Nucleotide sequence (SEQ ID NO: 1) and putative amino acid
sequence (SEQ ID NO: 2) of leucine-zipper protein kinase. Amino acid
numbering starts with the initiation codon.
FIGS. 2A(a)-2B(b). Northern blots of expression of leucine zipper protein
kinase in human tissue. FIGS. 2A(a)-2A(b) represent Northern blots
hybridized to .alpha.-[.sub.32 P] labeled leucine zipper protein kinase
cDNA from human adult tissue FIG. 2A(a) and from human fetal tissue FIG.
2A(b). FIGS. 2B(a)-2B(b) represent Northern blots hybridized to
.alpha.-[.sub.32 P] labeled .beta.-actin cDNA from human adult tissue FIG.
2B(a) and from human fetal tissue FIG. 2B(b).
DETAILED DESCRIPTION OF THE INVENTION
A novel member of the protein serine/threonine kinase family,
leucine-zipper protein kinase is provided by the present invention. As
used herein, the term leucine-zipper protein kinase (zpk) refers to a
protein having an amino acid sequence substantially homologous to at least
a portion of the amino acid sequence set forth in SEQ ID NO: 2. In
accordance with the present invention, the term "homologous" refers to a
one to one correlation between the sequences of two polypeptides or
oligonucleotides. Of course, 100% homology is not required in all cases.
In some instances polypeptides of the present invention may be
substantially homologous to the amino acid sequence set forth in SEQ ID
NO: 2. Substantial homology requires only that the essential nature of the
polypeptide, i.e. folding characteristics and unique features such as the
leucine zipper are preserved. Thus, modifications of the leucine-zipper
protein kinase are anticipated and are within the scope of the present
invention. These modification may be deliberate, as through site directed
mutagenesis, or may be accidental as through mutations in host which are
producers of the protein. In some embodiments of the present invention
polypeptides of the present invention may be at least about 75% homologous
to the sequence set forth in SEQ ID NO: 2. In other embodiments of the
present invention polypeptides may be at least about 85% homologous to the
sequence set forth in SEQ ID NO: 2. In yet other embodiments of the
present invention polypeptides may be at least about 95% homologous to the
sequence set forth in SEQ ID NO: 2. It is also anticipated that certain
non-commonly occurring amino acids may be substituted for commonly
occurring counterparts to confer desirable characteristics to the
resulting polypeptide.
Furthermore, it is contemplated in some aspects of the present invention
that a polypeptide may comprise only a portion of the sequence set forth
in SEQ ID NO: 2. This may be the case, for example, for a chimeric protein
encompassing active or otherwise desirable portions of a number of
proteins. A portion may also refer to a truncated polypeptide, be it
substantially truncated or only slightly truncated. Such truncated
polypeptides may be the result of an idiosyncracy in the mode of
production which results in truncation of amino acids from a terminal end,
or a finding that the truncated polypeptide works as well or better than
the full-length protein. For example, it might be found that the region
directly surrounding the protein kinase domain at amino acids 231-243 is
especially active.
Of course, in still other aspects of the present invention, the full-length
protein, as set forth in SEQ ID NO: 2, is contemplated.
The leucine-zipper protein kinase of the present invention, depending on
the pH of its environment, if suspended or in solution, or of its
environment when crystallized or precipitated, if in solid form, may be in
the form of pharmaceutically acceptable salts or may be in neutral form.
The free amino acid groups of the protein are, of course, capable of
forming acid addition salts with, for example, organic acids such as
hydrochloric, phosphoric, or sulfuric acid; or with organic acids such as,
for example, acetic, glycolic, succinic, or mandelic acid. The free
carboxyl groups are capable of forming salts with bases, including
inorganic bases such as sodium, potassium, or calcium hydroxides, and such
organic bases as piperidine, glucosamine, trimethylamine, choline, and
caffeine. In addition, the protein may be modified by combination with
other biological materials such as lipids and saccharides, or by side
chain modifications such as acetylation of amino groups, phosphorylation
of hydroxyl side chains or oxidation of sulfhydryl groups.
The leucine-zipper protein kinase is preferably purified and isolated.
"Purified" and "isolated" as the terms are used herein, are meant to refer
to molecules which have been purified or synthesized so as to be
substantially homogenous. The terms do not exclude the possibility that
certain impurities may be present in the composition as long as the
essential nature of the protein is intact.
Tissue distribution analysis indicated that leucine-zipper protein kinase
is present in the brain, more so in the adult than in the fetal brain
based upon the detection of a 3.4 Kb mRNA transcript. A smaller mRNA
transcript, about 3.2 Kb was detected in kidney and skeletal muscle. Adult
lung tissue expressed both transcripts at a very low level. In fetal
tissue, the only definite transcript seen is in the brain. These results
can be seen in FIGS. 2A(a)-2B(b).
The cDNA sequence of a novel leucine-zipper protein kinase is also provided
by the present invention. The cDNA has a long open reading frame encoding
668 amino acids. The methionine codon at nucleotides 99-101 matches
Kozak's consensus sequence for the initiation of translation. Kozak
Nucleic Acid Res 9: 5233-5252 (1981). The polyadenylation signal AATAAC
was found at nucleotides 3347-3352. Wickens and Stephenson, Science
226:1045-1051 (1984). The 5' cap site is CATCCG, 90 base pairs from the
initiation start site.
Homology searches of leucine-zipper protein kinase with the nucleotide and
amino acid databases showed no homology to any known protein kinase
family. Leucine-zipper protein kinase is most similar to serine/threonine
specific protein kinases. The leucine zipper protein kinase protein is
believed to be a "non-receptor type kinase" based on its lack of a
transmembrane domain. The consensus sequences for the ATP-binding site,
Gly-Xaa-Gly-Xaa-Xaa-Gly and Lys residues are found at positions 537-544
and 548, respectively. The protein kinase domain was found to be at
position 231-243. Taylor, et al., Annu. Rev. Cell Biol., 8, 429-462
(1992). At the C-terminus of the protein, there was two overlapping sites
of leucine zipper motif (leucine at every seventh amino acid), at position
442-468. A putative endoplasmic reticulum-targeting sequence was located
at residues 415-418 (Pelham, H. R. B., Annu. Rev. Cell Biol., 5, 1-23
(1989).
Comparison with other members of the family of protein kinases indicate
that leucine-zipper protein kinase has a number of novel features. First,
the glycine rich loop in leucine zipper protein kinase is present towards
the C-terminus of the catalytic domain, whereas in other protein kinases
it is present near the N-terminus.
Endoplasmic reticulum targeting sequences (REEL) have been identified in
both soluble; Pelham, H.R.B., Annu. Rev. Cell. Biol., 5, 1-23 (1989); and
transmembrane; Jackson, et al., EMBO J., 9, 3153-3162 (1990) endoplasmic
reticulum proteins. A lysine rich motif at the cytoplasmically-exposed
C-terminus of some transmembrane proteins was described which conferred
endoplasmic reticulum localization, although a more complex retention
signal at the C-terminus has also be postulated. Gabathuler and Kvist, J.
Cell Biol., 111, 1803-1810 (1990). Leucine-zipper protein kinase contains
an endoplasmic reticulum targeting sequence which is located from amino
acid 415-416, rather than at the extreme C-terminus of the protein.
Leucine-zipper protein kinase is also unique in that it contains a
leucine-zipper motif, a sequence in which leucines occur at every seventh
amino acid. Leucine-zippers contribute to targeting of various proteins
(eg. glucose transporters, Asano, et al., J. Biol. Chem., 267, 19636-19641
(1992)) and permit dimerization of various cytoplasmic hormone receptors
and enzymes. Forman, et al., Mol Endocrinol, 3, 1610-1626 (1989). Leucine
zippers are also a common feature of protein transcription factors, where
they permit homo- or heterodimerization resulting in tight binding to DNA
strands.
A leucine-zipper motif has been reported only once previously in a protein
kinase, the bovine cGMP-dependent protein kinase, which has a
leucine-isoleucine zipper motif at its N-terminus. Wernet, et al., FEBS,
251, 191-196 (1989).
Leucine-zipper protein kinase can be routinely synthesized in substantially
pure form by standard techniques well known in the art, such as
commercially available peptide synthesizers and the like.
Additionally, leucine-zipper protein kinase can be efficiently prepared
using any of numerous well known recombinant techniques such as those
described in U.S. Pat. No. 4,677,063 which patent is incorporated by
reference as if fully set forth herein. Briefly, most of the techniques
which are used to transform cells, construct vectors, extract messenger
RNA, prepare cDNA libraries, and the like are widely practiced in the art,
and most practitioners are familiar with the standard resource materials
which describe specific conditions and procedures. However, for
convenience, the following paragraphs may serve as a guideline.
Procaryotes most frequently are represented by various strains of E. coli.
However, other microbial strains may also be used, such as bacilli, for
example Bacillus subtilis, various species of Pseudomonas, or other
bacterial strains. In such procaryotic systems, plasmid vectors which
contain replication sites and control sequences derived from a species
compatible with the host are used. For example, E. coli is typically
transformed using derivatives of pBR322, a plasmid derived from an E. coli
species by Bolivar, et al, Gene (1977) 2:95. pBR322 contains genes for
ampicillin and tetracycline resistance, and thus provides additional
markers which can be either retained or destroyed in constructing the
desired vector. Commonly used procaryotic control sequences include
promoters for transcription initiation, optionally with an operator, along
with ribosome binding site sequences, include such commonly used promoters
as the beta-lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., Nature (1977) 198:1056) and the tryptophan (trp) promoter
system (Goeddel, et al. Nucleic Acids Res (1980) 8:4057) and the lambda
derived PL promoter and N-gene ribosome binding site (Shimatake, et al.,
Nature (1981) 92:128).
In addition to bacteria, eucaryotic microbes, such as yeast, may also be
used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's
yeast, are most used although a number of other strains are commonly
available. While vectors employing the 2 micron origin of replication are
illustrated, Broach, J. R., Meth Enz (1983) 101:307, other plasmid vectors
suitable for yeast expression are known (see, for example, Stinchcomb, et
al., Nature (1979) 282:39, Tschempe, et al., Gene (1980)10:157 and Clark,
L., et al., Meth Enz (1983) 101:300). Control sequences for yeast vectors
include promoters for the synthesis of glycolytic enzymes (Hess, et al., J
Adv Enzyme Req (1968) 7:149; Holland, et al. Biochemistry (1978) 17:4900).
Additional promoters known in the art include the promoter for
3-phosphoglycerate kinase (Hitzeman, et al., J Biol Chem (1980) 255:2073),
and those for other glycolytic enzymes such as glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other promoters, which have the additional advantage of transcription
controlled by growth conditions are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes
associated with nitrogen metabolism, and enzymes responsible for maltose
and galactose utilization (Holland, ibid). It is also believed terminator
sequences are desirable at the 3' end of the coding sequences. Such
terminators are found in the 3' untranslated region following the coding
sequences in yeast-derived genes. Many of the vectors illustrated contain
control sequences derived from the enolase gene containing plasmid peno46
(Holland, M. J., et al., J Biol Chem (1981) 256:1385) or the LEU2 gene
obtained from YEp13 (Broach, J., et al., Gene (1978) 8:121), however any
vector containing a yeast compatible promoter, origin of replication and
other control sequences is suitable.
It is also, of course, possible to express genes encoding polypeptides in
eucaryotic host cell cultures derived from multicellular organisms. See,
for example, Tissue Cultures, Academic Press, Cruz and Patterson, editors
(1973). Useful host cell lines include VERO, HeLa cells, and Chinese
hamster ovary (CHO) cells. Expression vectors for such cells ordinarily
include promoters and control sequences compatible with mammalian cells
such as, for example, the commonly used early and late promoters from
Simian Virus 40 (SV 40) Fiers, et al., Nature (1978) 273:113), or other
viral promoters such as those derived from polyoma, Adenovirus 2, bovine
papilloma virus, or avian sarcoma viruses. General aspects of mammalian
cell host system transformations have been described e.g. by Axel; U.S.
Pat. No. 4,399,216. It now appears, also that "enhancer" regions are
important in optimizing expression; these are, generally, sequences found
upstream or downstream of the promoter region in non-coding DNA regions.
Origins of replication may be obtained, if needed, from viral sources.
However, integration into the chromosome is a common mechanism for DNA
replication in eucaryotes. Plant cells are also now available as hosts,
and control sequences compatible with plant cells such as the nopaline
synthase promoter and polyadenylation signal sequences (Depicker, A., et
al., J Mol Appl Gen (1982) 1:561) are available.
Depending on the host cell used, transformation is done using standard
techniques appropriate to such cells. The calcium treatment employing
calcium chloride, as described by Cohen, S. N., Proc Natl Acad Sci (USA)
(1972) 69:2110, or methods described in Molecular Cloning: A Laboratory
Manual (1988) Cold Spring Harbor Press, could be used for procaryotes or
other cells which contain substantial cell wall barriers. Infection with
Agrobacterium tumefaciens (Shaw, C. H., et al., Gene (1983) 23:315) is
believed useful for certain plant cells. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham and van
der Eb, Virology (1978) 52:546 can be used. Transformations into yeast can
be carried out according to the method of Van Solingen, P., et al., J Bact
(1977) 130:946 and Hsiao, C. L., et al., Broc Natl Acad Sci (USA) (1979)
76:3829.
cDNA or genomic libraries can be screened using the colony hybridization
procedure. Generally, each microtiter plate is replicated onto duplicate
nitrocellulose filter papers (S&S type BA-85) and colonies are allowed to
grow at 37.degree. C. for 14-16 hr on L agar containing 50 .mu.g/ml Amp.
The colonies are lysed and DNA fixed to the filter by sequential treatment
for 5 min with 500 mM NaOH, 1.5M NaCl, and are washed twice for 5 min each
time with 5x standard saline citrate (SSC). Filters are air dried and
baked at 80.degree. C. for 2 hr. The duplicate filters are prehybridized
at 42.degree. C. for 6-8 hr with 10 ml per filter of DNA hybridization
buffer (5.times.SSC, pH 7.0 5.times. Denhardt's solution
(polyvinylpyrrolidine, plus Ficoll and bovine serum albumin;
1.times.=0.02% of each), 50 mM sodium phosphate buffer at pH 7.0, 0.2%
SDS, 20 .mu.g/ml Poly U, and 50 .mu.g/ml denatured salmon sperm DNA).
The samples can be hybridized with kinased probe under conditions which
depend on the stringency desired. Typical moderately stringent conditions
employ a temperature of 42.degree. C. for 24-36 hr with 1-5 ml/filter of
DNA hybridization buffer containing probe. For higher stringencies high
temperatures and shorter times are employed. Generally, the filters are
washed four times for 30 min each time at 37.degree. C. with 2.times.SSC,
0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then are washed twice
with 2xSSC and 0.2% SDS, air dried, and are autoradiographed
at--70.degree. C. for 2 to 3 days.
Construction of suitable vectors containing the desired coding and control
sequences employs standard ligation and restriction techniques which are
well understood in the art. Isolated plasmids, DNA sequences, or
synthesized oligonucleotides are cleaved, tailored, and religated in the
form desired.
Site specific DNA cleavage can be performed by treating the DNA with a
suitable restriction enzyme (or enzymes) under conditions which are
generally understood in the art, and the particulars of which are
specified by the manufacturer of these commercially available restriction
enzymes. See, e.g., New England Biolabs, Product Catalog. In general,
about 1 .mu.g of plasmid or DNA sequence is cleaved by one unit of enzyme
in about 20 .mu.l of buffer solution. Incubation times of about one hour
to two hours at about 37.degree. C. are workable, although variations can
be tolerated. After each incubation, protein can be removed by extraction
with phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol followed by running over a Sephadex G-5 spin column. If desired,
size separation of the cleaved fragments may be performed by
polyacrylamide gel or agarose gel electrophoresis using standard
techniques. A general description of size separations can be found in
Methods in Enzymology (1980) 65:499-560.
Restriction cleaved fragments may be blunt ended by treating with the large
fragment of E. coli DNA polymerase I (Klenow) in the presence of the four
deoxynucleotide triphosphates (dNTPs) using incubation times of about 15
to 25 min at 20.degree. to 25.degree. C. in 50 mM Tris pH 7.6, 50 mM NaCl,
6 mM MgCl.sub.2, 6 mM DTT and 5-10 .mu.M dNTPs. The Klenow fragment fills
in at 5' sticky ends but chews back protruding 3' single strands, even
though the four dNTPs are present. If desired, selective repair can be
performed by supplying only one of the, or selected, dNTPs within the
limitations dictated by the nature of the sticky ends. After treatment
with Klenow, the mixture is extracted with phenol/chloroform and ethanol
precipitated followed by running over a Sephadex G-50 spin column.
Treatment under appropriate conditions with S1 nuclease results in
hydrolysis of any single-stranded portion.
Synthetic oligonucleotides can be prepared by the triester method of
Metteucci, et al. (J Am Chem Soc (1981) 103:3185) or using commercially
available automated oligonucleotide synthesizers. Kinasing of single
strands prior to annealing or for labeling is achieved using an excess,
e.g., approximately 10 units of polynucleotide kinase to 0.1 nmole
substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl.sub.2, 5 mM
dithiothreitol, 1-2 Mm ATP, 1.7 pmoles .gamma..sup.32 P-ATP (2.9
mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
Ligations can be performed in 15-30 .mu.l volumes under the following
standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM
MgCl.sub.2, 10 mM DTT, 33 .mu.g/ml GSA, 10 mM-50 mM NaCl, and either 40
.mu.M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0.degree. C. (for
"sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at
14.degree. C. (for "blunt end" ligation). Intermolecular "sticky end"
ligations are usually performed at 33-100 .mu.g/ml total DNA
concentrations (5-100 nM total end concentration). Intermolecular blunt
end ligations (usually employing a 10-30 fold molar excess of linkers) are
performed at 1 .mu.M total ends concentration.
In vector construction employing "vector fragments", the vector fragment
can be treated with bacterial alkaline phosphatase (BAP) in order to
remove the 5' phosphate and prevent religation of the vector. BAP
digestions can be conducted at pH 8 in approximately 150 mM Tris, in the
presence of Na+and Mg+.sup.2 using about 1 unit of BAP per .mu.g of vector
at 60.degree. C. for about one hour. In order to recover the nucleic acid
fragments, the preparation is extracted with phenol/chloroform and ethanol
precipitated and desalted by application to a Sephadex G-50 spin column.
Alternatively, religation can be prevented in vectors which have been
double digested by additional restriction enzyme digestion of the unwanted
fragments.
For portions of vectors derived from cDNA or genomic DNA which require
sequence modifications, site specific primer directed mutagenesis can be
used. This is conducted using a primer synthetic oligonucleotide
complementary to a single stranded phage DNA to be mutagenized except for
limited mismatching, representing the desired mutation. Briefly, the
synthetic oligonucleotide is used as a primer to direct synthesis of a
strand complementary to the phage, and the resulting double-stranded DNA
is transformed into a phage-supporting host bacterium. Cultures of the
transformed bacteria are plated in top agar, permitting plaque formation
from single cells which harbor the phage.
Theoretically, 50% of the new plaques will contain the phage having, as a
single strand, the mutated form; 50% will have the original sequence. The
resulting plaques can be hybridized with kinased synthetic primer at a
temperature which permits hybridization of an exact match, but at which
the mismatches with the original strand are sufficient to prevent
hybridization. Plaques which hybridize with the probe are then picked,
cultured, and the DNA recovered.
Correct ligations for plasmid construction can be confirmed by first
transforming a suitable host with the ligation mixture. Successful
transformants are selected by ampicillin, tetracycline or other antibiotic
resistance or using other markers depending on the mode of plasmid
construction, as is understood in the art. Plasmids from the transformants
can then be prepared according to the method of Clewell, D. B., et al.
Proc Natl Acad Sci (USA) (1969) 62:1159, optionally following
chloramphenicol amplification (Clewell, D. B., J Bacteriol (1972)
110:667). The isolated DNA Is analyzed by restriction and/or sequenced by
the dideoxy method of Snager, F., et al. Proc Natl Acad Sci (USA) (1977)
74:5463 as further described by Messing, et al., F. Supp. Nucleic Acids
Res (1981) 9.309, or by the method of Maxam, et al., Methods in Enzymology
(1980) 65:499.
In accordance with the present invention polynucleotide probes specifically
hybridizable to a portion of the leucine zipper protein kinase gene are
provided. Polynucleotide probes substantially homologous to a portion of
the leucine-zipper protein kinase gene are also provided. Such probes may
be used for diagnostic or research purposes to detect or quantitate the
expression of leucine zipper protein kinase in a sample such as by
detecting the presence or absence of polynucleotide duplex formation
between the polynucleotide probe and leucine-zipper protein kinase gene.
Samples may derived from cell culture or may be derived from a patient.
Samples may be biological fluids such as synovial fluid in some aspects of
the invention. Tissue samples may also be used in some embodiments of the
present invention. Detection of the presence of polynucleotide duplexes is
indicative of the presence of the leucine-zipper protein kinase gene in a
sample and may be indicative of diseases associated with leucine zipper
protein kinase, such as tumors of the central nervous system. Provision of
means for detecting hybridization of polynucleotides with the
leucine-zipper protein kinase gene can routinely be accomplished. Such
provision may include enzyme conjugation, radiolabelling or any other
suitable detection systems. Kits for detecting the presence or absence of
leucine zipper protein kinase or a particular transcript thereof may also
be prepared. Said polynucleotide probes may range in length from about 5
to about 100 nucleotide units. In more preferred embodiments of the
present invention the probes may be from about 8 to about 75 nucleotide
units in length. Ideally, said probes range in length from about 12 to
about 50 nucleotide units. It is recognized that since polynucleotide
probes of the present invention may preferably not exceed 100 nucleotides
in length, said probes may specifically hybridize to only a portion of the
targeted sequence. The portion of the leucine zipper protein kinase
sequence to be targeted can be identified by one skilled in the art. Most
suitably, a target sequence is chosen which is unique, thereby decreasing
background noise attributable to hybridization by the probe other than to
the target. By way of example, one skilled in the art would be unlikely to
select a repeating sequence of adenine nucleotide units as this is a
common sequence occurring in many genes. The practitioner might choose to
perform a search and comparison of sequences found in a sequence
repository such as Genbank in order to identify and design a useful probe.
Such methods of conventionally used to identify unique sequences. These
unique sequences, when used as probes, need not necessarily be crucial to
the regulation of the expression of leucine-zipper protein kinase.
In accordance with other methods of the present invention, neuronal cells
may be contacted with leucine-zipper protein kinase, or a portion thereof
in order to inhibit cellular proliferation. While not wishing to be bound
to a particular theory, it is believed that the addition of exogenous
leucine-zipper protein kinase, or portions thereof may interfere with
specific protein-protein or protein-nucleic acid interactions involved in
cellular hyperproliferation. For example, by administering an inactive
leucine-zipper protein kinase polypeptide or a portion thereof, it may be
possible to compete with naturally occurring leucine-zipper protein kinase
for binding regions of target nucleic acid molecules or polypeptides in
order to modulate its effect in the cell at the level of protein-protein
or protein-nucleic acid interactions. In this way, it may be possible to
treat a mammal suffering from tumors of the central nervous system by
inhibiting the overexpression of leucine-zipper protein kinase in vivo or
by interfering with a vital signal in the chain of signals leading to
tumorigenicity.
For methods of the present invention, leucine-zipper protein kinase may be
formulated into pharmacological compositions containing an effective
amount of leucine-zipper protein kinase and a usual nontoxic carrier, such
carriers being known to those skilled in the art. The compositions may be
administered by a method suited to the form of the composition. Such
compositions are, for example, in the form of usual liquid preparations
including solutions, suspensions, emulsions, and the like which can be
given orally, intravenously, subcutaneously or intramuscularly.
The present invention is also directed to methods of inhibiting
hyperproliferation of neuronal cells comprising contacting the cells with
oligonucleotides substantially complementary to a portion of the nucleic
acid sequence set forth in SEQ ID NO: 1. "Complementary" in the context of
this invention, means the ability to form hydrogen bonds, also known as
Watson-Crick base pairing, between complementary bases, usually on
opposite nucleic acid strands or two regions of a nucleic acid strand, to
form a double-stranded duplex. Guanine and cytosine are examples of
complementary bases which are known to form three hydrogen bonds between
them. Adenine and thymine are examples of complementary bases which are
known to form two hydrogen bonds between them. "Specifically hybridizable"
and "substantially complementary" are terms which indicate a sufficient
degree of complementarity to avoid non-specific binding of the
oligonucleotide (or polynucleotide probe) to non-target sequences under
conditions in which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays and therapeutic treatment, or, in
the case of in vitro assays, under conditions in which the assays are
conducted. It is understood that an oligonucleotide or polynucleotide
probe need not be 100% complementary to its target nucleic acid sequence
to be specifically hybridizable or effective in methods of the present
invention. In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic
acid. This term includes oligomers consisting of naturally occurring
bases, sugars and intersugar (backbone) linkages as well as oligomers
having non-naturally occurring portions which function similarly. The
oligonucleotides in accordance with this invention preferably comprise
from about 5 to about 50 nucleotide units. It is more preferred that such
oligonucleotides comprise from about 8 to 30 nucleotide units, and still
more preferred to have from about 12 to 25 nucleotide units.
Oligonucleotides of the present invention may be prepared by standard
techniques such as solid-phase synthesis which are well known to those
skilled in the art.
Furthermore, in accordance with methods of the present invention, a
therapeutically effective amount of oligonucleotide is administered to a
mammal suffering from tumors of the central nervous system.
Oligonucleotides may be formulated in a pharmaceutical composition, which
may include carriers, thickeners, diluents, buffers, preservatives,
surface active agents and the like in addition to the oligonucleotide.
Pharmaceutical compositions may also include one or more active
ingredients such as antimicrobial agents, antiinflammatory agents,
anesthetics, and the like in addition to oligonucleotides.
The pharmaceutical compositions of the present invention may be
administered in a number of ways depending on whether local or systemic
treatment is desired, and on the area to be treated. Administration may be
done topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example by
intravenous drip or subcutaneous, intraperitoneal or intramuscular
injection.
Formulations for topical administration may include ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases,
thickeners and the like may be necessary or desirable. Coated condoms or
gloves may also be useful.
Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, capsules, sachets,
or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids
or binders may be desirable.
Formulations for parenteral administration may include sterile aqueous
solutions which may also contain buffers, diluents and other suitable
additives.
Dosing is dependent on severity and responsiveness of the condition to be
treated, but will normally be one or more doses per day, with course of
treatment lasting from several days to several months or until a cure is
effected or a diminution of disease state is achieved. Persons of ordinary
skill can easily determine optimum dosages, dosing methodologies and
repetition rates.
The following examples are illustrative and are not meant to be limiting of
the present invention.
EXAMPLES
EXAMPLE 1
Cells
Human teratocarcinoma line NT2 was differentiated into postmitotic neurons
NT2-N with retinoic acid as previously described (Pleasure et al., J.
Neurosci 12: 1802-1815 (1992); Younkin et al., Proc. Natl. Acad. Sci. USA
90: 2174-2178 (1992)). Poly(A).sup.+ RNA was isolated from both NT2 and
NT2-N neurons using Invitrogen mRNA kit.
EXAMPLE 2
Subtractive Hybridization and DNA Amplification
Invitrogen's Subtractor probe kit was used according to the manufacturer's
instructions to isolate two different subtracted cDNAs UND and DIFF. UND
was enriched in transcripts expressed in the undifferentiated stage
whereas DIFF was enriched in transcripts present in the neurons. One .mu.g
portions of UND and DIFF mRNA were used for PCT amplification with
degenerate primers as described in Wilks Proc. Natl. Acad. Sci. USA 86:
1603-1607 (1989). PCT was performed using a Geneamp kit (Cetus) with 1
.mu.g of each of the degenerate primers. The final concentration of
magnesium was 2.1 mM. PCT cycling was performed on a Perkin-Elmer 480
thermal cycler for 39 cycles with a profile of 1.3 minutes at 95.degree.
C. (denaturation), 2 minutes at 45.degree. C. (annealing), and 2 minutes
at 64.degree. C. (elongation).
EXAMPLE 3
Subcloning of Amplified DNAs and DNA Sequencing
The PCR reaction mixture were run on 4% Nusieve agarose gel and the
amplified band of .about.220 bp was excised. The band was purified using
Magic PCR Kit (Promega). The amplified DNA was digested with the
restriction enzymes BamH1/EcoR1. The amplified DNAs were subcloned into
the BamH1 and EcoR1 cleaved Bluescript DNA. A total of about 200 clones
(100 representing UND and 100 representing DIFF) were examined by
sequencing using a Taq DyeDeoxy terminator cycle sequencing kit (Applied
Biosystems). Plasmid DNA was isolated using Qiagen column 20. The cycle
sequencing reactions were performed in a Perkin-Elmer 480 thermal cycler
for 25 cycles with a profile of 96.degree. C. for 30 seconds, 40.degree.
C. for 15 seconds, and 60.degree. C. for 4 minutes. Following separation
of the extension products on a Select-D G-50 column (5 Prime 3 Prime) the
reaction mixtures were dried, resuspended in 4 .mu.l of 5:1 formamide/50
mM EDTA, loaded on a 6% sequencing gel, and analyzed using an Applied
Biosystems 373 fluorescent sequencer.
EXAMPLE 4
cDNA Library Screening
The 210 bp 10.2 PCR clone from undifferentiated clones was radiolabelled
with [.sup.32 P]dCTP and used to probe .about.10.sup.6 plaques from an
amplified human fetal brain library (Stratagene) to obtain larger cDNA
clones. Hybridization was carried out overnight at 42.degree. C. in 50%
Formamide, 5xSSPE, 5xDenhardt's, 1% SDS, 100ug/ml sheared salmon sperm
DNA, and 1.times.10.sup.6 cpm/ml of probe. Filters were washed at
60.degree. C. twice in 2xSSC containing 0.1% SDS, and exposed overnight to
Kodak XAR-5 film at -70.degree. C.
EXAMPLE 5
Sequence Determination
cDNAs were subcloned into a plasmid vector BluescriptSk. For complete
sequence determination, unidirectional nested deletions was performed
using the Exo111/Mung Bean nuclease kit from Stratagene. The colonies
obtained after deletions were sequenced as described earlier using a Taq
DyeDeoxy terminator cycle sequencing kit (Applied Biosystems). The DNA
sequence obtained was determined after sequencing twice.
EXAMPLE 6
Sequence Comparisons
All sequence manipulations were done on a VAX using the University of
Wisconsin Genetics Computer Group Sequence Analysis Software Package. DNA
fragments obtained after nested deletions was assembled into Contigs using
the Programme Sequencer 2.0 (Gene Codes Corp). Protein analysis was done
using MacVector (IBI).
EXAMPLE 7
RNA Analysis
Human multiple tissue northern blots were purchased from Clontech
laboratories. Hybridization conditions were similar to that used for
library screening. Filters were washed to a final stringency of 0.1
.times.SSC/0.1% SDS at 65.degree. C. before exposure to XAR-5 x-ray film.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 2
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3426 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 99..2105
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AGCATCCGGAGCGGAGCTGCAGCAGCGCCGCCTTTTGTGCTGCGGCCGCGGAGCCCCCGA60
GGGCCCAGTGTTCACCATCATACCAGGGGCCAGAGGCGATGGCTTGCCTCCAT113
MetAlaCysLeuHis
15
GAGACCCGAACACCCTCTCCTTCCTTTGGGGGCTTTGTGTCTACCCTA161
GluThrArgThrProSerProSerPheGlyGlyPheValSerThrLeu
101520
AGTGAGGCATCCATGCGCAAGCTGGACCCAGACACTTCTGACTGCACT209
SerGluAlaSerMetArgLysLeuAspProAspThrSerAspCysThr
253035
CCCGAGAAGGACCTGACGCCTACCCATGTCCTGCAGCTACATGAGCAG257
ProGluLysAspLeuThrProThrHisValLeuGlnLeuHisGluGln
404550
GATGCAGGGGGCCCAGGGGGAGCAGCTGGGTCACCTGAGAGTCGGGCA305
AspAlaGlyGlyProGlyGlyAlaAlaGlySerProGluSerArgAla
556065
TCCAGAGTTCGAGCTGACGAGGTGCGACTGCAGTGCCAGAGTGGCAGT353
SerArgValArgAlaAspGluValArgLeuGlnCysGlnSerGlySer
70758085
GGCTTCCTTGAGGGCCTCTTTGGCTGCCTGCGCCCTGTCTGGACCATG401
GlyPheLeuGluGlyLeuPheGlyCysLeuArgProValTrpThrMet
9095100
ATTGGCAAAGCCTACTCCACTGAGCACAAGCAGCAGCAGGAAGACCTT449
IleGlyLysAlaTyrSerThrGluHisLysGlnGlnGlnGluAspLeu
105110115
TGGGAGGTCCCCTTTGAGGAAATCCTGGACCTGCAGTGGGTGGGCTCA497
TrpGluValProPheGluGluIleLeuAspLeuGlnTrpValGlySer
120125130
GGGGCCCAGGGTGCTGTCTTCCTGGGGCGCTTCCACGGGGAGGAGGTG545
GlyAlaGlnGlyAlaValPheLeuGlyArgPheHisGlyGluGluVal
135140145
GCTGTGAAGAAGGTGCGAGACCTCAAAGAAACCGACATCAAGCACTTG593
AlaValLysLysValArgAspLeuLysGluThrAspIleLysHisLeu
150155160165
CGAAAGCTGAAGCACCCCAACATCATCACTTTCAAGGGTGTGTGCACC641
ArgLysLeuLysHisProAsnIleIleThrPheLysGlyValCysThr
170175180
CAGGCTCCCTGCTACTGCATCCTCATGGAGTTCTGCGCCCAGGGCCAG689
GlnAlaProCysTyrCysIleLeuMetGluPheCysAlaGlnGlyGln
185190195
CTGTATGAGGTACTGCGGGCTGGCCGCCCTGTCACCCCCTCCTTACTG737
LeuTyrGluValLeuArgAlaGlyArgProValThrProSerLeuLeu
200205210
GTTGACTGGTCCATGGGCATCGCTGGTGGCATGAACTACCTGCACCTG785
ValAspTrpSerMetGlyIleAlaGlyGlyMetAsnTyrLeuHisLeu
215220225
CACAAGATTATCCACAGGGATCTCAAGTCACCCAACATGCTAATCACC833
HisLysIleIleHisArgAspLeuLysSerProAsnMetLeuIleThr
230235240245
TACGACGATGTGGTGAAGATCTCAGATTTTGGCACTTCCAAGGAGCTG881
TyrAspAspValValLysIleSerAspPheGlyThrSerLysGluLeu
250255260
AGTGACAAGAGCACCAAGATGTCCTTTGCAGGGACAGTAGCCTGGATG929
SerAspLysSerThrLysMetSerPheAlaGlyThrValAlaTrpMet
265270275
GCCCCTGAGGTGATCCGCAATGAACCTGTGTCTGAGAAGGTCGACATC977
AlaProGluValIleArgAsnGluProValSerGluLysValAspIle
280285290
TGGTCCTTTGGCGTGGTGCTATGGGAACTGCTGACTGGTGAGATCCCC1025
TrpSerPheGlyValValLeuTrpGluLeuLeuThrGlyGluIlePro
295300305
TACAAAGACGTAGATTCCTCAGCCATTATCTGGGGTGTGGGAAGCAAC1073
TyrLysAspValAspSerSerAlaIleIleTrpGlyValGlySerAsn
310315320325
AGTCTCCATCTGCCCGTGCCCTCCAGTTGCCCAGATGGTTTCAAGATC1121
SerLeuHisLeuProValProSerSerCysProAspGlyPheLysIle
330335340
CTGCTTCGCCAGTGCTGGAATAGCAAACCACGAAATCGCCCATCATTC1169
LeuLeuArgGlnCysTrpAsnSerLysProArgAsnArgProSerPhe
345350355
CGACAGATCCTGCTGCATCTGGACATTGCCTCAGCTGATGTACTCTCC1217
ArgGlnIleLeuLeuHisLeuAspIleAlaSerAlaAspValLeuSer
360365370
ACACCCCAGGAGACTTACTTTAAGTCCCAGGCAGAGTGGCGGGAAGAA1265
ThrProGlnGluThrTyrPheLysSerGlnAlaGluTrpArgGluGlu
375380385
GTAAAACTGCACTTTGAAAAGATTAAGTCAGAAGGGACCTGTCTGCAC1313
ValLysLeuHisPheGluLysIleLysSerGluGlyThrCysLeuHis
390395400405
CGCCTAGAAGAGGAACTGGTGATGAGGAGGAGGGAGGAGCTCAGACAC1361
ArgLeuGluGluGluLeuValMetArgArgArgGluGluLeuArgHis
410415420
GCCCTGGACATCAGGGAGCACTATGAAAGGAAGCTGGAGAGAGCCAAC1409
AlaLeuAspIleArgGluHisTyrGluArgLysLeuGluArgAlaAsn
425430435
AACCTGTATATGGAACTTAATGCCCTCATGTTGCAGCTGGAACTCAAG1457
AsnLeuTyrMetGluLeuAsnAlaLeuMetLeuGlnLeuGluLeuLys
440445450
GAGAGGGAGCTGCTCAGGCGAGAGCAAGCTTTAGAGCGGAGGTGCCCA1505
GluArgGluLeuLeuArgArgGluGlnAlaLeuGluArgArgCysPro
455460465
GGCCTGCTGAAGCCACACCCTTCCCGGGGCCTCCTGCATGGAAACACA1553
GlyLeuLeuLysProHisProSerArgGlyLeuLeuHisGlyAsnThr
470475480485
ATGGAGAAGCTTATCAAGAAGAGGAATGTGCCACAGAATCTGTCACCC1601
MetGluLysLeuIleLysLysArgAsnValProGlnAsnLeuSerPro
490495500
CATAGCCAAAGGCCAGATATCCTCAAGGCGGAGTCTTTGCTCCCTAAA1649
HisSerGlnArgProAspIleLeuLysAlaGluSerLeuLeuProLys
505510515
CTAGATGCAGCCCTGAGTGGGGTGGGGCTTCCTGGGTGTCCTAAGGCC1697
LeuAspAlaAlaLeuSerGlyValGlyLeuProGlyCysProLysAla
520525530
CCCCCCTCACCAGGACGGAGTCGCCGTGGCAAGACCCGTCACCGCAAG1745
ProProSerProGlyArgSerArgArgGlyLysThrArgHisArgLys
535540545
GCCAGCGCCAAGGGGAGCTGTGGGGACCTGCCTGGGCTTCGTACAGCT1793
AlaSerAlaLysGlySerCysGlyAspLeuProGlyLeuArgThrAla
550555560565
GTGCCACCCCATGAACCTGGAGGACCAGGAAGCCCAGGGGGCCTAGGA1841
ValProProHisGluProGlyGlyProGlySerProGlyGlyLeuGly
570575580
GGGGGACCCTCAGCCTGGGAGGCCTGCCCTCCCGCCCTCCGTGGGCTT1889
GlyGlyProSerAlaTrpGluAlaCysProProAlaLeuArgGlyLeu
585590595
CATCATGACCTCCTGCTCCGCAAAATGTCTTCATCGTCCCCAGACCTG1937
HisHisAspLeuLeuLeuArgLysMetSerSerSerSerProAspLeu
600605610
CTGTCAGCAGCACTAGGGTCCCGGGGCCGGGGGGCCACAGGCGGAGCT1985
LeuSerAlaAlaLeuGlySerArgGlyArgGlyAlaThrGlyGlyAla
615620625
GGGGATCCTGGCTCACCACCTCCGGCCCGGGGTGACACCCCACCAAGT2033
GlyAspProGlySerProProProAlaArgGlyAspThrProProSer
630635640645
GAGGGCTCACCCCCTGGCTCCACCAGCCCAGATTCACCTGGGGAGCCA2081
GluGlySerProProGlySerThrSerProAspSerProGlyGluPro
650655660
AAGGGGAACCACCTCCTCCAGTAGGGCCTGGTGAAGGTGTGGGGCTTCTGG2132
LysGlyAsnHisLeuLeuGln
665
GAACTGGAAGGGAAGGGACCTCAGGCCGGGGAGGAAGCCGGGCTGGGTCCCAGCACTTGA2192
CCCCATCTGCACTGCTGTACAGGGCTGCCGTCACCCGAAGTCAGAAACGTGGCATCTCAT2252
CGGAAGAGGAGGAAGGAGAGGTAGACAGTGAAGTAGAGCTGACATCAAGCCAGAGGTGGC2312
CTCAGAGCCTGAACATGCGCCAGTCACTATCTACCTTCAGCTCAGAGAATCCATCAGATG2372
GGGAGGAAGGCACAGCTAGTGAACCTTCCCCCAGTGGCACACCTGAAGTTGGCAGCACCA2432
ACACTGATGAGCGGCCAGATGAGCGGTCTGATGACATGTGCTCCCAGGGCTCAGAAATCC2492
CACTGGACCCACCTCCTTCAGAGGTCATCCCTGGCCCTGAACCCAGCTCCCTGCCCATTC2552
CACACCAGGAACTTCTCAGAGAGCGGGGCCCTCCCAATTCTGAGGACTCAGACTGTGACA2612
GCACTGAATTGGACAACTCCAACAGCGTTGATGCCTTGCGCCCCCCAGCTTCCCTCCCTC2672
CATGAAAGCCACTCGTATTCCTTGTACATAGAGAAATATTTATATGGATTATATATATAT2732
ACATATATATATATATATGCGCCACATAATCAACAGAAAGATGGGGCTGTCCCAGCCGTA2792
AGTCAGGCTCGAGGGAGACTGATCCCCTGACCAATTCACCTGATAAACTCTAGGGACACT2852
GGCAGCTGTGGAAATGAATGAGGCACAGCCGTAGAGCTGTGGCTAAGGGCAAGCCCCTTC2912
CTGCCCCACCCCATTCCTTATATTCAGCAAGCAACAAGGCAATAGAAAAGCCAGGGTTGT2972
CTTTATATTCTTTATCCCCAAATAATAGGGGGTGGGGGGAGGGGCGGTGGGAGGGGCAGG3032
AGAGAAAACCACTTAGACTGCACTTTTCTGTTCCGTTTACTCTGTTTACACATTTTGCAC3092
TTGGGAGGAGGGAGGCTAAGGCTGGGTCCTCCCCTCTGAGGTTTCTCAGGTGGCAATGTA3152
ACTCATTTTTTTGTCCCACCATTTATCTTCTCTGCCCAAGCCCTGTCTTAAGGCCCAGGG3212
GGAGGTTAGGAGACTGATAGCATGTGATGGCTCAGGCTGAAGAACCGGGGTTCTGTTTAA3272
GTCCCTGCTTTTATCCTGGTGCCTGATTGGGGTGGGGACTGTCCTACTGTAACCCCTGTG3332
AAAAACCTTGAAAAATAACACTCCATGCAGGAAAAAAAAAAAAAAAAAAAAAAAAAGGAA3392
TTCGATATCAAGCTTATCGATACCGTCGACCTCG3426
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 668 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetAlaCysLeuHisGluThrArgThrProSerProSerPheGlyGly
151015
PheValSerThrLeuSerGluAlaSerMetArgLysLeuAspProAsp
202530
ThrSerAspCysThrProGluLysAspLeuThrProThrHisValLeu
354045
GlnLeuHisGluGlnAspAlaGlyGlyProGlyGlyAlaAlaGlySer
505560
ProGluSerArgAlaSerArgValArgAlaAspGluValArgLeuGln
65707580
CysGlnSerGlySerGlyPheLeuGluGlyLeuPheGlyCysLeuArg
859095
ProValTrpThrMetIleGlyLysAlaTyrSerThrGluHisLysGln
100105110
GlnGlnGluAspLeuTrpGluValProPheGluGluIleLeuAspLeu
115120125
GlnTrpValGlySerGlyAlaGlnGlyAlaValPheLeuGlyArgPhe
130135140
HisGlyGluGluValAlaValLysLysValArgAspLeuLysGluThr
145150155160
AspIleLysHisLeuArgLysLeuLysHisProAsnIleIleThrPhe
165170175
LysGlyValCysThrGlnAlaProCysTyrCysIleLeuMetGluPhe
180185190
CysAlaGlnGlyGlnLeuTyrGluValLeuArgAlaGlyArgProVal
195200205
ThrProSerLeuLeuValAspTrpSerMetGlyIleAlaGlyGlyMet
210215220
AsnTyrLeuHisLeuHisLysIleIleHisArgAspLeuLysSerPro
225230235240
AsnMetLeuIleThrTyrAspAspValValLysIleSerAspPheGly
245250255
ThrSerLysGluLeuSerAspLysSerThrLysMetSerPheAlaGly
260265270
ThrValAlaTrpMetAlaProGluValIleArgAsnGluProValSer
275280285
GluLysValAspIleTrpSerPheGlyValValLeuTrpGluLeuLeu
290295300
ThrGlyGluIleProTyrLysAspValAspSerSerAlaIleIleTrp
305310315320
GlyValGlySerAsnSerLeuHisLeuProValProSerSerCysPro
325330335
AspGlyPheLysIleLeuLeuArgGlnCysTrpAsnSerLysProArg
340345350
AsnArgProSerPheArgGlnIleLeuLeuHisLeuAspIleAlaSer
355360365
AlaAspValLeuSerThrProGlnGluThrTyrPheLysSerGlnAla
370375380
GluTrpArgGluGluValLysLeuHisPheGluLysIleLysSerGlu
385390395400
GlyThrCysLeuHisArgLeuGluGluGluLeuValMetArgArgArg
405410415
GluGluLeuArgHisAlaLeuAspIleArgGluHisTyrGluArgLys
420425430
LeuGluArgAlaAsnAsnLeuTyrMetGluLeuAsnAlaLeuMetLeu
435440445
GlnLeuGluLeuLysGluArgGluLeuLeuArgArgGluGlnAlaLeu
450455460
GluArgArgCysProGlyLeuLeuLysProHisProSerArgGlyLeu
465470475480
LeuHisGlyAsnThrMetGluLysLeuIleLysLysArgAsnValPro
485490495
GlnAsnLeuSerProHisSerGlnArgProAspIleLeuLysAlaGlu
500505510
SerLeuLeuProLysLeuAspAlaAlaLeuSerGlyValGlyLeuPro
515520525
GlyCysProLysAlaProProSerProGlyArgSerArgArgGlyLys
530535540
ThrArgHisArgLysAlaSerAlaLysGlySerCysGlyAspLeuPro
545550555560
GlyLeuArgThrAlaValProProHisGluProGlyGlyProGlySer
565570575
ProGlyGlyLeuGlyGlyGlyProSerAlaTrpGluAlaCysProPro
580585590
AlaLeuArgGlyLeuHisHisAspLeuLeuLeuArgLysMetSerSer
595600605
SerSerProAspLeuLeuSerAlaAlaLeuGlySerArgGlyArgGly
610615620
AlaThrGlyGlyAlaGlyAspProGlySerProProProAlaArgGly
625630635640
AspThrProProSerGluGlySerProProGlySerThrSerProAsp
645650655
SerProGlyGluProLysGlyAsnHisLeuLeuGln
660665
__________________________________________________________________________
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